PROJECT 3 - Abstract This proposal will determine the influence of tick gut microbiota on tick gut immune signaling pathways JAK/STAT and IMD and the functional consequence on Borrelia burgdorferi, and Anaplasma phagocytophilum survival in the tick - an important determinant of infection prevalence. Our earlier work (Narasimhan et al, Cell Host Microbe 15:58-71, 2014) and our recent work (Abraham et al, Proc Natl Acad Sci,114: E781-790, 2017) demonstrate that tick gut microbiota modulates B. burgdorferi colonization and A. phagocytophilum infection of Ixodes scapularis. Perturbation of the gut microbiota (dysbiosis) disrupts the integrity of the peritrophic matrix - an acellular glycoprotein-rich layer that separates the gut epithelium from the lumen. While B. burgdorferi resides on the gut epithelium and an intact peritrophic matrix (PM) affords protection from toxic components in the tick gut enhancing their colonization of the gut, A. phagocytophilum circumvents the PM barrier to infect the gut and finally the salivary glands by as-yet-unknown mechanisms and exploits a compromised PM to efficiently breach the PM barrier. These observations reveal a novel and apparently contrasting facet of Borrelia-tick and Anaplasma-tick interactions. In a more recent effort (Narasimhan et al, Nat Commun. 184: s41467, 2017), we draw attention to the dynamic interactions of the tick microbiota with tick gut defense responses and to the diverse ways in which tick microbiota might influence pathogen survival in the tick gut. These findings, together with the observations of Projects 1 and 2 emphasize the influence of the interactions between tick gut microbiota- and immune responses on pathogen survival in the tick. In Aim 1 we will characterize the extent to which gut microbiota interacts with tick gut immune responses (specifically JAK/STAT and IMD) and host cytokines (specifically IFN?) that enter the tick gut, and define gut bacterial profiles that are significantly influenced by the tick and host immune responses and vice-versa. In Aim 2 we will reconstitute specific gut microbiota or bacterial components and determine how this influences tick gut immune signaling and ultimately the survival of B. burgdorferi or A. phagocytophilum in the tick This research endeavor will unfold a mechanistic understanding of the functional consequence of tick gut microbiota on tick gut immune signaling and the survival of two important tick-borne pathogens that cause human disease.